Methods for inhibiting angiogenesis

ABSTRACT

A method of inhibiting angiogenesis is disclosed comprising administering to a human in need thereof an effective amount of a compound having the formula ##STR1## wherein R 1  and R 3  are independently hydrogen, --CH 3 , ##STR2##  wherein Ar is optionally substituted phenyl; R 2  is selected from the group consisting of pyrrolidine and piperidino; or a pharmaceutically acceptable salt of solvate thereof.

This application is a continuation of application Ser. No. 08/137,703, filed on Oct. 15, 1993, now abandoned.

BACKGROUND OF THE INVENTION

Angiogenesis (or neovascularization), the development of blood vessels, plays an important role in embryonic development, in the inflammatory response, in the development of metastases (tumor induced angiogenesis or TIA), in diabetic retinopathy, in the formation of the arthritic panus and in psoriasis. In tumor angiogenesis, for example, capillary sprouts are formed, their formation being induced by a group of tumor cells. These sprouts eventually develop into a microcirculatory network within the tumor mass. There are two principle types of tumor angiogenesis in terms of the events which follow implantations of metastatic seedlings on surfaces and in organs.

The first or primary angiogenesis is the initial vascularization of the mass of multiplying tumor cells and is regarded as an essential prerequisite for the survival and further growth of a metastatic deposit.

The second is a continuing or secondary angiogenesis and is the phenomenon which occurs in waves at the periphery of a growing tumor mass. This second angiogenesis is essential for the accretion of new microcirculatory territories into the service of the expanding and infiltrating tumor.

It is apparent that a chemical which would inhibit angiogenesis, either by competitively inhibiting an angiogenesis factor or by some other mechanism, would have an adverse effect upon the growth of tumors, on the development of retinopathy or rheumatoid arthritis, or on the development of the psoriatic lesion.

SUMMARY OF THE INVENTION

This invention provides methods for inhibiting angiogenesis and/or angiogenic diseases, comprising administering to a human in need of treatment an effective amount of a compound of formula I ##STR3## wherein R¹ and R³ are independently hydrogen, --CH₃, ##STR4## wherein Ar is optionally substituted phenyl; R² is selected from the group consisting of pyrrolidino and piperidino; and pharmaceutically acceptable salts and solvates thereof.

DETAILED DESCRIPTION OF THE INVENTION

The current invention concerns the discovery that a select group of 2-phenyl-3-aroylbenzothiophenes (benzothiophenes), those of formula I, are useful for inhibiting angiogenesis and/or angiogenic diseases. The methods of treatment provided by this invention are practiced by administering to a human in need of a dose of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof, that is effective to inhibit angiogenesis and/or angiogenic diseases. The term inhibit is defined to include its generally accepted meaning which includes prophylactically treating a human subject to incurring angiogenesis and/or angiogenic diseases, and holding in check and/or treating existing angiogenesis and/or angiogenic diseases. As such, the present method includes both medical therapeutic and/or prophylactic treatment, as appropriate.

Generally, the compound is formulated with common excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for convenient oral administration, or administered by the intramuscular or intravenous routes. The compounds can be administered transdermally, and may be formulated as sustained release dosage forms and the like.

The compounds used in the methods of the current invention can be made according to established procedures, such as those detailed in U.S. Pat. Nos. 4,133,814, 4,418,068, and 4,380,635 all of which are incorporated by reference herein. In general, the process starts with a benzo[b]thiophene having a 6-hydroxyl group and a 2-(4-hydroxyphenyl) group. The starting compound is protected, alkylated, and deprotected to form the formula I compounds. Examples of the preparation of such compounds are provided in the U.S. patents discussed above. Substituted phenyl includes phenyl substituted once or twice with C₁ -C₆ alkyl, C₁ -C₄ alkoxy, hydroxy, nitro, chloro, fluoro, or tri(chloro or fluoro)methyl.

The compounds used in the methods of this invention form pharmaceutically acceptable acid and base addition salts with a wide variety of organic and inorganic acids and bases and include the physiologically acceptable salts which are often used in pharmaceutical chemistry. Such salts are also part of this invention. Typical inorganic acids used to form such salts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, phosphoric, hypophosphoric and the like. Salts derived from organic acids, such as aliphatic mono and dicarboxylic acids, phenyl substituted alkanoic acids, hydroxyalkanoic and hydroxyalkandioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, may also be used. Such pharmaceutically acceptable salts thus include acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate, phenylbutyrate, B-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate, caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate, glycollate, heptanoate, hippurate, lactate, malate, maleate, hydroxymaleate, malonate, mandelate, mesylate, nicotinate, isonicotinate, nitrate, oxalate, phthalate, teraphthalate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, propiolate, propionate, phenylpropionate, salicylate, sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate, chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, methanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate, and the like. A preferred salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formed by reacting a compound of formula I with an equimolar or excess amount of acid. The reactants are generally combined in a mutual solvent such as diethyl ether or benzene. The salt normally precipitates out of solution within about one hour to 10 days and can be isolated by filtration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of salts include ammonium hydroxide and alkali and alkaline earth metal hydroxides, carbonates and bicarbonates, as well as aliphatic and primary, secondary and tertiary amines, aliphatic diamines and hydroxy alkylamines. Bases especially useful in the preparation of addition salts include ammonium hydroxide, potassium carbonate, sodium bicarbonate, calcium hydroxide, methylamine, diethylamine, ethylene diamine, cyclohexylamine and ethanolamine.

The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.

Pharmaceutical formulations can be prepared by procedures known in the art. For example, the compounds can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. Examples of excipients, diluents, and carriers that are suitable for such formulations include the following: fillers and extenders such as starch, sugars, mannitol, and silicic derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin, and polyvinyl pyrrolidone; moisturizing agents such as glycerol; disintegrating agents such as calcium carbonate and sodium bicarbonate; agents for retarding dissolution such as paraffin; resorption accelerators such as quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorptive carriers such as kaolin and bentonite; and lubricants such as talc, calcium and magnesium stearate, and solid polyethyl glycols.

The compounds can also be formulated as elixirs or solutions for convenient oral administration or as solutions appropriate for parenteral administration, for instance by intramuscular, subcutaneous or intravenous routes. Additionally, the compounds are well suited to formulation as sustained release dosage forms and the like. The formulations can be so constituted that they release the active ingredient only or preferably in a particular part of the intestinal tract, possibly over a period of time. The coatings, envelopes, and protective matrices may be made, for example, from polymeric substances or waxes.

The particular dosage of a compound of formula I required to inhibit angiogenesis and/or angiogenic diseases, according to this invention will depend upon the severity of the condition, the route of administration, and related factors that will be decided by the attending physician. Generally, accepted and effective daily doses will be from about 0.1 to about 1000 mg/day, and more typically from about 50 to about 200 mg/day. Such dosages will be administered to a subject in need of treatment from once to about three times each day, or more often as needed to effectively inhibit angiogenesis and/or angiogenic diseases.

It is usually preferred to administer a compound of formula I in the form of an acid addition salt, as is customary in the administration of pharmaceuticals bearing a basic group, such as the piperidino ring. It is also advantageous to administer such a compound by the oral route to an aging human. For such purposes the following oral dosage forms are available.

Formulations

In the formulations which follow, "Active ingredient" means a compound of formula I.

Formulation 1: Gelatin Capsules

Hard gelatin capsules are prepared using the following:

    ______________________________________                                         Ingredient        Quantity (mg/capsule)                                        ______________________________________                                         Active ingredient 0.1-1000                                                     Starch, NF        0-650                                                        Starch flowable powder                                                                           0-650                                                        Silicone fluid 350 centistokes                                                                   0-15                                                         ______________________________________                                    

The ingredients are blended, passed through a No. 45 mesh U.S. sieve, and filled into hard gelatin capsules.

Examples of specific capsule formulations of the compound of formula 1 wherein R² is piperidino, (raloxifene), that have been made include those shown below:

Formulation 2: Raloxifene capsule

    ______________________________________                                         Ingredient        Quantity (mg/capsule)                                        ______________________________________                                         Raloxifene        1                                                            Starch, NF        112                                                          Starch flowable powder                                                                           225.3                                                        Silicone fluid 350 centistokes                                                                   1.7                                                          ______________________________________                                    

Formulation 3: Raloxifene capsule

    ______________________________________                                         Ingredient        Quantity (mg/capsule)                                        ______________________________________                                         Raloxifene        5                                                            Starch, NF        108                                                          Starch flowable powder                                                                           225.3                                                        Silicone fluid 350 centistokes                                                                   1.7                                                          ______________________________________                                    

Formulation 4: Raloxifene capsule

    ______________________________________                                         Ingredient        Quantity (mg/capsule)                                        ______________________________________                                         Raloxifene        10                                                           Starch, NF        103                                                          Starch flowable powder                                                                           225.3                                                        Silicone fluid 350 centistokes                                                                   1.7                                                          ______________________________________                                    

Formulation 5: Raloxifene capsule

    ______________________________________                                         Ingredient        Quantity (mg/capsule)                                        ______________________________________                                         Raloxifene        50                                                           Starch, NF        150                                                          Starch flowable powder                                                                           397                                                          Silicone fluid 350 centistokes                                                                   3.0                                                          ______________________________________                                    

The specific formulations above may be changed in compliance with the reasonable variations provided.

A tablet formulation is prepared using the ingredients below:

Formulation 6: Tablets

    ______________________________________                                         Ingredient       Quantity (mg/tablet)                                          ______________________________________                                         Active ingredient                                                                               0.1-1000                                                      Cellulose, microcrystalline                                                                     0-650                                                         Silicon dioxide, fumed                                                                          0-650                                                         Stearate acid    0-15                                                          ______________________________________                                    

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.1-1000 mg of active ingredient are made up as follows:

Formulation 7: Tablets

    ______________________________________                                         Ingredient          Quantity (mg/tablet)                                       ______________________________________                                         Active ingredient   0.1-1000                                                   Starch              45                                                         Cellulose, microcrystalline                                                                        35                                                         Polyvinylpyrrolidone                                                                               4                                                          (as 10% solution in water)                                                     Sodium carboxymethyl cellulose                                                                     4.5                                                        Magnesium stearate  0.5                                                        Talc                1                                                          ______________________________________                                    

The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50°-60° C. and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.

Suspensions each containing 0.1-1000 mg of medicament per 5 mL dose are made as follows:

Formulation 8: Suspensions

    ______________________________________                                         Ingredient          Quantity (mg/5 ml)                                         ______________________________________                                         Active ingredient   0.1-1000   mg                                              Sodium carboxymethyl cellulose                                                                     50         mg                                              Syrup               1.25       mg                                              Benzoic acid solution                                                                              0.10       mL                                              Flavor              q.v.                                                       Color               q.v.                                                       Purified water to   5          mL                                              ______________________________________                                    

The medicament is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form a smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.

TEST PROCEDURE

Primary retinal capillary endothelial cell cultures are prepared in substantial accordance with the procedure described in Buzney et al., Retinal Vascular Endothelial Cell and Pericytes: Differential Growth Characteristics in vitro, Invest. Ophthalmol. Visual Sci. 24: 470-483 (1983) and are separated from contaminating cells as described in Voyta et al., Identification and Isolation of Endothelial Cells Based on their Increased uptake of Acetylated-low density Lipoprotein, J. Cell Biol. 99: 2034-2040 (1981).

Alternatively, bovine heart endothelial cells are obtained (American Type Culture Collection; Rockville Md. 20852) for use in the assay. Irrespective of source,the endothelial cells are seeded into 24 well tissue culture plates at a cell density of 10,000 cells/well in Dulbecco's modified Eagle Medium (GIBCO; Grand Island, N.Y.) supplemented with 10% bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 20 ng/ml fibroblast growth factor. After overnight incubation, various amounts of the compounds of the invention are added to individual growth wells. For comparison, native PF-4, known to inhibit enthothelial cells and angiogenesis, is tested over a range of 0.1 to 4 μg/ml, served as a positive control. The endothelial cell cultures are allowed to grow for four days at 37° C, 5% CO₂ in a humidified tissue culture incubator. The cultures are then harvested individually by trypsinization and counted using a Am-Coulter Counter (Coulter Electronics, Inc.; Opa Locka Fla. 33054).

Activity in the above test indicates the compounds of the invention are of potential in the treatment of angiogenesis and/or angiogenic diseases. 

We claim:
 1. A method of inhibiting angiogenesis comprising administering to a human in need thereof an effective amount of a compound having the formula ##STR5## wherein R¹ and R³ are independently hydrogen, --CH₃, ##STR6## wherein Ar is optionally substituted phenyl; R² is selected from the group consisting of pyrrolidine and piperidino; or a pharmaceutically acceptable salt or solvate thereof.
 2. The method of claim 1 wherein said compound is the hydrochloride salt thereof.
 3. The method of claim 1 wherein said administration is prophylactic.
 4. The method of claim 1 wherein said compound is ##STR7## or its hydrochloride salt. 